The present invention is directed towards a process for the amplification of a target nucleic acid sequence contained in a larger nucleic acid independent of using a thermocycle or a thermostable polymerase. Unlike current technologies that employ a thermocycle and are therefore dependent upon a thermostable polymerase, the current invention allows for specific primer template association at a low temperature that will remain constant over the duration of the reaction.
A method for the site-specific amplification of a region of nucleic acid is described. Current amplification technology is based upon the Polymerase Chain Reaction (PCR). This PCR system can be thought of as involving three main components. First DNA oligonucleotides complementary to the flanking ends of the target sequence are required. These DNA oligonucleotides serve as primers for the initiation of DNA replication by the second component of the system, a thermostable DNA dependent DNA polymerase, such as the Taq polymerase. The use of a thermostable DNA polymerase is absolutely required in PCR so that the polymerase activity can survive the third component, the thermal cycle. The thermal cycle uses high temperatures, usually 95 degrees Celsius, to melt the target duplex DNA so that a subsequent annealing temperature, usually in the range of 50-60 degrees Celsius, permits the annealing of the primers to the appropriate locations flanking the target DNA. Following the annealing step, the thermal cycle incorporates a polymerization temperature, usually 72 degrees Celsius, which is the optimal temperature of polymerization for the current thermostable polymerases used in PCR.
The requirement of a thermal cycle to facilitate the annealing of the primers flanking the target DNA to be amplified has several drawbacks. Primer annealing temperature is an important parameter in the success of PCR amplification. The annealing temperature is characteristic for each oligonucleotide: it is a function of the length and base composition of the primer as well as the ionic strength of the reaction buffer. The theoretical amplification value is never achieved in practice. Several factors prevent this from occurring, including: competition of complementary daughter strands with primers for reannealing (i.e. two daughter strands reannealing results in no amplification); loss of enzyme activity due to thermal denaturation, especially in the later cycles; even without thermal denaturation, the amount of enzyme becomes limiting due to molar target excess in later cycles (i.e. after 25-30 cycles too many primers need extending); possible second site primer annealing and non-productive priming. Moreover, primers must avoid stretches of polybase sequences (e.g. poly dG) or repeating motifs—these can hybridize with inappropriate register on the template. Inverted repeat sequences should also be avoided so as to prevent formation of secondary structure in the primer, which would prevent hybridization to template.
An additional drawback is the costly need for temperature baths, which are required to shift their temperatures up and down rapidly, and in an automated programmed manner. These are known as thermal cyclers or PCR machines.
A further problem with PCR is the lack of fidelity of the various Polymerases (Table 1) under different conditions. However, with increasing number of cycles the greater the probability of generating various artifacts (e.g. mispriming products). It is unusual to find procedures that have more than 40 cycles. Errors made by DNA polymerase can affect the extension. reaction of PCR during five distinct steps: (1) the binding of the correct dNTP by polymerase; (2) the rate of phosphodiester bond formation; (3) the rate of pyrophosphate release; (4) the continuation of extension after misincorporation; and (5) the ability of the enzyme to adjust to a misincorporated base by providing 3′-to-5′ exonuclease ‘proofreading’ activity. Misincorporation rates for different polymerases are described in terms of errors per nucleotide polymerized, and the rate can be greatly affected by many parameters. Several studies have concluded that different thermostable DNA polymerases have error rates as high as 2.1×10−4 to 1.6×10−6 errors per nucleotide per extension (Table 2).
Another major drawback is that standard PCR protocols can amplify DNA sequences of 3000 base pairs (3 kb) or less. Efficient long PCR requires the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3′ to 5′ exo) is present at a lower concentration. Following the results of Cheng et al. the Tth enzyme (ABI/Perkin-Elmer) enzyme has been used as the main-component polymerase and Vent (New England Biolabs) as the fractional-component polymerase. Other combinations of proofreading and non-proofreading polymerases are difficult to employ because different activities in specific buffer systems limits which combinations of polymerases can be used. Moreover, all of the problems associated with standard PCR reactions become even more critical when attempting to amplify regions of DNA 3 kb or longer.
The current invention eliminates these problems with traditional PCR by eliminating the need for a thermal cycle and a thermostable polymerase in the amplification of a sequence of DNA embedded within a longer target DNA. The current invention replaces the thermal cycle required to anneal the primers to the flanking ends of a target template by utilizing the enzymes active during homologous recombination, more specifically during homologous pairing or D-loop formation.
In bacteriophage T4, DNA replication, as well as being initiated from specific origins of replication, is also very efficiently initiated from recombination intermediates. Therefore; the current invention is directed at a system that primes DNA replication, in a specific manner, via recombination intermediates formed at opposite ends of a target sequence embedded within a much larger sequence. This permits the reaction to be run at room temperature and therefore permits the use of a non-thermal stable polymerase, The primary advantage of employing a non thermostable polymerase is that several polymerases have been characterized which have far superior fidelity. Moreover, the characterization of accessory factors, such as sliding clamp proteins, are known to increase the length of DNA which can be amplified to entire genomes. In addition, the utilization of enzymes to deliver the primers eliminates all of the problems associated with annealing primers within the context of a thermal cycle mentioned above. Moreover, the homologous pairing reaction catalyzed by the bacteriophage T4 proteins is extremely efficient and would eliminate the problem of mis-priming.
TABLE 1Thermostable DNA polymerases and their sourcesDNA PolymeraseNatural or recombinantSourceTaqNaturalThermus aquaticusAmplitaq ®RecombinantT. aquaticusAmplitaq (StoffelRecombinantT. aquaticusfragment) ®Hot Tub ™NaturalThermus flavisPyrostase ™NaturalT. flavisVent ™RecombinantThermococcus litoralisDeep Vent ™RecombinantPyrococcus GB-DTthRecombinantThermus thermophilusPfuNaturalPyrococcus furiosusULTma ™RecombinantThermotoga maritima
TABLE 2Properties of DNA polymerases used in PCRTaq/StoffelDeepAmplitaq ®fragmentVent ™Vent ™PfuTthULTma ™95° C. half-40 min80 min400 min1380 min>120 min20 min>50 minlife5′3′ exo+−−−−+−3′5′ exo−−+++−+Extension75>50>80?60>33?rate (nt/sec)RT activityWeakWeak???Yes?Resulting3′ A3′ A>95%>95%blunt3′ AbluntendsbluntbluntStrand−−++−−−displacementM.W. (kDa)94  61??92  9470Error Rates    1. Taq (Thermus aquaticus)
1.1×10−4 base substitutions/bp [Tindall, K. R., and Kunkel, T. A. (1988) Biochemistry 27, p6008-6013, “Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase.”]
2.4×10−5 frameshift mutations/bp [Tindall and Kunkel, Id.]
2.1×10−4 errors/bp [Keohavong, P., and Thilly, W. G. (1989) Proc Natl Acad Sci USA 86(23), p9253-9257, “Fidelity of DNA polymerases in DNA amplification.”]
7.2×10−5 errors/bp [Ling, L. L., Keohavong, P., Dias, C., and Thilly, W. G. (1991) PCR Methods Appl 1(1) p63-69, “Optimization of the polymerase chain reaction with regard to fidelity: modified T7, Taq, and Vent DNA polymerases.”]
8.9×10−5 errors/bp [Cariello, N. F., Swenberg, J. A., and Skopek, T. R. (1991) Nucleic Acids Res 19(15), p4193-4198, “Fidelity of Thermococcus Litoralis DNA Polymerase (Vent) in PCR determined by denaturing gradient gel electrophoresis.”]
2.0×10−5 errors/bp [Lundberg, K. S., Shoemaker, D. D., Adams, M. W., Short, J. M., Sorge, J. A., and Mathur, E. J. (1991) Gene 108(1), p1-6, “High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus.”]
1.1×10−4 errors/bp [Barnes, W. M. (1992) Gene 112(1), p29-35, “The Fidelity of Taq polymerase catalyzing PCR is improved by an N-terminal deletion.”]    2. KlenTaq (Thermus aquaticus, N-terminal deletion mutant)
5.1×10−5 errors/bp [Barnes, W. M. (1992) Gene 112(1), p29-35, “The Fidelity of Taq polymerase catalyzing PCR is improved by an N-terminal deletion.”]    3. Vent (Thermococcus litoralis)
2.4×10−5 errors/bp [Cariello, N. F., Swenberg, J. A., and Skopek, T. R. (1991) Nucleic Acids Res 19(15), p4193-4198, “Fidelity of Thermococcus Litoralis DNA Polymerase (Vent) in PCR determined by denaturing gradient gel electrophoresis.”]
4.5×10−5 errors/bp [Ling, L. L., Keohavong, P., Dias, C., and Thilly, W. G. (1991) PCR Methods Appl 1(1) p63-69, “Optimization of the polymerase chain reaction with regard to fidelity: modified T7, Taq, and Vent DNA polymerases.”]
5.7×10-5 errors/bp [Matilla, P., Korpela, J., Tenkanen, T., and Pitkanen, K. (1991) Nucleic Acids Res 19(18), p4967-4973, “Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase—an extremely heat stable enzyme with proofreading activity.”]    4. Vent(exo-) (Thermococcus litoralis)
1.9×10−4 errors/bp [Matilla et al., Id.]    5. Deep Vent (Pyrococcus species GB-D)
No published literature. New England Biolabs claims fidelity is equal to or greater than that of Vent.    6. Deep Vent(exo-)
No published literature.    7. Pfu (Pyrococcus furiosus)
1.6×10−6 errors/base [Lundberg, K. S., Shoemaker, D. D., Adams, M. W., Short, J. M., Sorge, J. A., and Mathur, E. J. (1991) Gene 108(1), p1-6, “High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus.”]    8. Replinase (Thermus flavis)
1.03×10−4 errors/base [Matilla, P., Korpela, J., Tenkanen, T., and Pitkanen, K. (1991) Nucleic Acids Res 19(18), p4967-4973, “Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase—an extremely heat stable enzyme with proofreading activity.”]